In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted, as reflected or fluorescent light, from the sample. The focus of an illuminating light beam is moved in a sample plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detection light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a diaphragm (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen with the focus of the illuminating light beam, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scanning module that is flange-mounted onto the stand of a conventional light microscope, the scanning module containing all the aforesaid elements additionally necessary for scanning a sample.
Commercial scanning microscopes usually contain a microscope stand such as the one also used in conventional light microscopy. Confocal scanning microscopes, in particular, are usually also usable as conventional light microscopes. In conventional fluorescent incident-light microscopy, the component of the light of a light source, for example an arc lamp, that contains the desired wavelength region for fluorescent excitation is coupled therefrom into the microscope beam path with the aid of a color filter (called the “excitation filter”). Incoupling into the beam path of the microscope is accomplished by means of a dichroic beam splitter that reflects the excitation light to the sample while allowing the fluorescent light proceeding from the sample to pass largely unimpeded. The excitation light backscattered from the sample is held back with a blocking filter that is nevertheless transparent to the fluorescent radiation. It has been usual for some time to optimally combine mutually coordinated filters and beam splitters into an easily exchangeable modular filter block. The filter blocks are usually arranged in a revolving magazine within the microscope as part of so-called fluorescent incident illuminators, thus making possible rapid and easy exchange.
In confocal scanning microscopy, a detection pinhole can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light to be detected therefore very probably originates almost exclusively from the focus region, rendering superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
A non-descan arrangement, in which the detection light does not travel to the detector via the beam deflection device (descan arrangement) and the beam splitter for incoupling the illuminating light, but instead is deflected directly to the objective by means of a dichroic beam splitter and detected, is of interest in particular given that the fluorescent photon yield with two-photon excitation is in any case low, since less light is usually lost along this detection light pathway. In addition, scattered components of the detection light make a substantial contribution to the signal in the case of two-photon excitation with descan detection, whereas these play a much lesser role in non-descan detection. Arrangements of this kind are known, for example, from the publication of David W. Piston et al., “Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity,” Applied Optics, Vol. 33, No. 4, February 1996; and from Piston et al., “Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy,” SPIE, Vol. 1640.
U.S. Pat. No. 6,169,289 B1 discloses a microscope with multi-photon excitation in which the detection light proceeding from the sample is detected on the condenser side.
One problem of the known arrangements for non-descan detection is the fact that, the beam splitter for deflecting the detection light out of the microscope beam path occupies a great deal of installation space, especially along the microscope beam path, so that it often cannot be accommodated in ordinary microscope stands. Massive physical modifications to the scanning microscope, and in particular to the microscope stand, are often necessary in order to achieve nonscan detection.